Steady-state study of the mechanism of dopa-oxidase activity of tyrosinase.

The mechanism of the dopa-oxidase activity of frog epidermis tyrosinase has been studied. Initial reaction rates have been measured as function of substrate concentrations, L-dopa and oxygen, in the presence and absence of an inhibitor, product of the reaction. Initial reaction rates versus substrate concentrations, without inhibitor, show a linear dependence in the double-reciprocal space, that discarded Ordered and Random mechanisms. Initial reaction rates versus substrate concentrations, in the presence of an inhibitor product of the reaction, show a non-linear dependence in the double-reciprocal space. This point, joined to the former one, indicates a Ping-Pong mechanism, different of the Hexa-Uni type. The reaction is discussed for first time taking into account a trisubstrate mechanism. The experimental results lead to an (Uni Uni Bi Uni) Ping-Pong mechanism. On the other hand, they can explain the differences between known data of tyrosinases from several sources. Michaelis constant have been calculated for both substrates. The values are 0.16 and 7.14 mM for oxygen and L-dopa respectively.